No abstract

No abstract

No abstract

Chinese hamster ovary (CHO) cells are one of the most important cell lines in biological research, and are the most widely used host for industrial production of recombinant therapeutic proteins. Despite their extensive applications, little sequence information is available for molecular based research. To facilitate gene discovery and genetic engineering, two cDNA libraries were constructed from three CHO cell lines grown under various conditions. The average insert size for both libraries is approximately 800-850 bp, and each library has comparable redundancy levels of 36%-38% for the sequences isolated. Random sequencing of 4,608 ESTs yielded 2,602 unique assemblies, 76% of which were annotated as orthologs of sequences in the GenBank database. A high abundance of mitochondrial genome transcripts facilitated the assembly of the complete mitochondrial genome by PCR walking. Comparative analysis of sequences from both mitochondrial and nuclear genomes with orthologous genes from other species shows that CHO sequences are generally most similar to mouse; however, examples with highest similarity to rat or human are common. A cDNA microarray, including all 4,608 ESTs, was constructed. The microarray results reveal a high level of consistency between transcript abundance in the libraries and fluorescence intensities. Inclusion of redundant clones in the microarray, additionally, allows small changes in abundant mRNAs to be discerned with a high degree of confidence. The information and tools generated provide access to genomic technology for this important cell line.

No abstract

DNA microarray technology has been widely utilized for species with extensive genome sequence information available. Given the limited genomic information pertaining to Chinese hamster ovary (CHO) cell line, cross-species hybridization using mouse microarrays provides a viable alternative. In this study, the utility of mouse Affymetrix microarrays for transcriptome profiling in CHO cells was assessed by hybridizing identical sets of cRNA samples from CHO cells on both mouse and CHO Affymetrix microarrays. Expression level measured by probe sets for orthologous transcripts on the two microarrays was compared. Only a fraction of the orthologous probes which detected expression calls in same species hybridization were similarly called present in cross species hybridization. In further analysis at the 25-mer probe level, it was revealed that specific hybridization signals were detectable by the subset of mouse probes that have a high degree of homology to the corresponding CHO sequences. The feasibility of cross species hybridization for quantifying the extent of differential expression was assessed by comparing transcript levels of CHO cells cultivated with and without sodium butyrate. While same species hybridization gave consistent degree of differential expression calls in replicated runs, a much inferior ability in quantifying differential expression was seen with cross species hybridization. Our results demonstrate that through detailed analysis of homology at the probe pair level, a subset of probes on existing mouse Affymetrix oligo-array can be used successfully for transcriptome profiling of CHO cells.

Chinese hamster ovary (CHO) cells are a prevalent tool in biological research and are among the most widely used host cell lines for production of recombinant therapeutic proteins. While research in other organisms has been revolutionized through the development of DNA sequence-based tools, the lack of comparable genomic resources for the Chinese hamster has impeded similar work in CHO cell lines. A comparative genomics approach, based upon the completely sequenced mouse genome, can facilitate genomic work in this important organism. Using chromosome synteny to define regions of conserved linkage between Chinese hamster and mouse chromosomes, a working scaffold for the Chinese hamster genome has been developed. Mapping CHO and Chinese hamster sequences to the mouse genome creates direct access to relevant information in public databases. Additionally, mapping gene expression data onto a chromosome scaffold affords the ability to interpret information in a genomic context, potentially revealing important structural and regulatory features in the Chinese hamster genome. Further development of this genomic scaffold will provide opportunities to use biomolecular tools for research in CHO cell lines today and will be an asset to future efforts to sequence the Chinese hamster genome.

Genomic and proteomic based global gene expression profiling has altered the landscape of biological research in the past few years. Its potential impact on cell culture bioprocessing has only begun to emanate, partly due to the lack of genomic sequence information for the most widely used industrial cells, Chinese hamster ovary (CHO) cells. Transcriptome and proteome profiling work for species lacking extensive genomic resources must rely on information for other related species or on data obtained from expressed sequence tag (EST) sequencing projects, for which burgeoning efforts have only recently begun. This article discusses the aspects of EST sequencing in those industrially important, genomic resources-poor cell lines, articulates some of the unique features in employing microarray in the study of cultured cells, and highlights the infrastructural needs in establishing a platform for genomics based cell culture research. Recent experience has revealed that generally, most changes in culture conditions only elicit a moderate level of alteration in gene expression. Nevertheless, by broadening the conventional scope of microarray analysis to consider estimated levels of transcript abundance, much physiological insight can be gained. Examples of the application of microarray in cell culture are discussed, and the utility of pattern identification and process diagnosis are highlighted. As genomic resources continue to expand, the power of genomic tools in cell culture processing research will be amply evident. The key to harnessing the immense benefit of these genomic resources resides in the development of physiological understanding from their application.